Helicobacter suis (H. suis) DNA in gastric biopsies compared to control patients. H. suis is a zoonotic Helicobacter species that naturally colonizes the stomach of pigs and non-human primates but can be transmitted to humans. Here, we investigated the influence of a gastric H. suis infection on PD disease progression through a 6-hydroxydopamine (6-OHDA) mouse model. Therefore, mice with either a short- or long-term H. suis infection were stereotactically injected with 6-OHDA in the left striatum and sampled one week later. Remarkably, a reduced loss of dopaminergic neurons was seen in the H. suis/6-OHDA groups compared to the control/6-OHDA groups. Correspondingly, motor function of the H. suis-infected 6-OHDA mice was superior to that in the non-infected 6-OHDA mice. Interestingly, we also observed higher expression levels of antioxidant genes in brain tissue from H. suis-infected 6-OHDA mice, as a potential explanation for the reduced 6-OHDA-induced cell loss. Our data support an unexpected neuroprotective effect of gastric H. suis on PD pathology, mediated through changes in oxidative stress.The exact etiology of Parkinson’s disease (PD) remains largely unknown, but more and more research suggests the involvement of the gut microbiota. Interestingly, idiopathic PD patients were shown to have at least a 10 times higher prevalence of  Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease, affecting more than 6 million people worldwide in 2016 and exceeding 12–17 million by 2040 ,4. PD clHelicobacter (H.) pylori infection has already been associated to an increased risk of developing PD, and also with worse motor function in established PD [H. pylori infection may affect the bioavailability of levodopa, which is used for PD treatment, resulting in worse motor control in H. pylori-infected PD patients [H. pylori eradication has been suggested to result in improvement of PD-related motor symptoms [There is increasing evidence that PD might be initiated in the gastrointestinal tract ,12,13,14ished PD ,19,20,21patients . H. pylosymptoms ,24,25,26symptoms .H. suis DNA was found in gastric biopsies from patients with idiopathic PD compared to a non-PD control group (27% vs 2%) [H. suis infection in a PD patient resulted in a remarkable improvement of the gastric as well as the neurological symptoms [H. suis is a zoonotic spiral-shaped non-H. pylori Helicobacter (NHPH) species naturally colonizing the stomach of pigs and non-human primates [H. suis-infected pigs and humans. Raw or undercooked pork may also constitute a source of H. suis infection in humans, as H. suis can be present and survive in minced pork [H. suis to cause extra-gastric effects was confirmed in a recent mouse study from our lab [H. suis infection of 1 month was shown to not only induce gastric inflammation, but also to cause a disturbance of the gastrointestinal barrier integrity, low-grade systemic inflammation, disruption of the blood–cerebrospinal fluid (CSF) barrier, and brain inflammation accompanied by cognitive decline [H. suis infection in neurodegenerative disorders, and PD in particular, requires further investigation.Interestingly, in one study, an exceptional high frequency of % vs 2%) . In addisymptoms . H. suisprimates . Transmiced pork . The abi our lab . In this decline . ClearlyThe 6-hydroxydopamine (6-OHDA) model is often used as an experimental model for PD as it leads to its main pathological and clinical hallmarks . The modH. suis in PD, short-term and long-term H. suis-infected mice were subjected to a unilateral injection with 6-OHDA in the left striatum. One week later, the effects of H. suis infection on PD-related hallmarks were evaluated.To further investigate the potential role of H. suis in the corpus and antrum of the stomach was confirmed after sampling, in both the short- and long-term infected mice  were demonstrated (data not shown). We observed no significant changes in expression levels of inflammatory genes that were tested in the antrum of the stomach in the infected mice (data not shown).The experimental set-up is depicted in ted mice A,B. In te medium A,B. Analted mice , indicatted mice C,D was mnfection E–H, whilMuc13, might contribute to loss of epithelial cell polarity and cell–cell interactions [Muc13 in the stomach corpus in the long-term but not in the short-term infection group  in the stomach were not significantly altered (data not shown). Tight junctions, consisting of both transmembrane and cytosolic proteins, are crucial elements for maintaining cell-to-cell adhesion of the epithelial cells underneath this mucus layer [Cldn5) and zonula occludens 1 (Zo1) were significantly decreased in the corpus of long-term infected mice (Zo1 and Zo3 was demonstrated in the corpus (Ocln), claudin 1 (Cldn1), and claudin 3 (Cldn3) were seen (data not shown). In both groups there were no significant changes in tight junction expression levels in the antrum of the stomach (data not shown). The gastrointestinal barrier integrity was also assessed by measuring the level of fluorescence in the plasma 5 h after intragastric FITC-dextran gavage. While a small increase in fluorescence was seen in the plasma of long-term H. suis-infected mice, no significant changes were demonstrated  A,B, the H. suis/6-OHDA and control/6-OHDA subgroups.Similarly, in the pole test of both groups at 7 dpi C,D, the H. suis/6-OHDA subgroup. For the long-term group, the stride length of both the control/6-OHDA and H. suis/6-OHDA subgroups were shorter compared to the other subgroups as well, albeit not significantly.PD patients tend to show a shuffling gait pattern with a shortened stride length . TherefoH. suis/6-OHDA subgroup compared to the other subgroups, albeit not significantly. For the long-term group at 7 dpi  of the right, non-injected striatum was set at 100% , and the TH signal in the left, injected striatum was then calculated with the conversion factor. For the short-term group, the mean % loss of dopaminergic neuron fiber density in the left striatum compared to the right striatum was 93.17% for the control/6-OHDA subgroup, and 77.78% for the subgroup A. As expsubgroup B. In FigH. suis/6-OHDA subgroups, and this was significant between the H. suis/6-OHDA and the H. suis/vehicle subgroups, as well as between the control/6-OHDA and control/vehicle subgroup. The number of activated microglial cells was similar in the left and right striatum of both vehicle-injected subgroups. We also performed an immunostaining to visualize reactive astrocytes using GFAP, which was automatically analyzed and calculated in a similar way as the TH and the IBA1 staining , and this was analyzed by manual counting A for thestaining B. For alNrf2), which has also been specifically implicated in PD pathogenesis [The loss of dopaminergic neurons in the substantia nigra and the intraneuronal aggregation of alpha-synuclein are the main pathological hallmarks of PD, which are both mechanistically linked to oxidative stress ,47. For ogenesis ,49,50. Iogenesis . Sod1 and Sod2) convert superoxide radicals to form H2O2, which is subsequently detoxified by catalase (Cat) and glutathione peroxidase (Gpx1 and Gpx2) [Sod2 showed a significant increase for the 6-OHDA-injected H. suis short-term infected group with a similar trend in the long-term group  . The exprm group A,B, no sup , NADPH oxidases (Nox1 and Nox2), thioredoxins , glutathione synthesizing enzymes such as glutathione-disulfide reductase (Gsr) and glutamate-cysteine ligase  and a light regulatory subunit (Gclm)) with glutathione being the principal redox buffer in tissues. For the long-term infected mice, we observed a significant increase in gene expression for Gclc, Gclm, Gsr, Hmox1, and Nox2 in parallel to the changes seen in Nrf2 when comparing the H. suis/6-OHDA mice with the control/6-OHDA mice  and long-term infection (17 months), the H. suis/6-OHDA subgroup performed better than the control/6-OHDA subgroup, especially in the traversal beam and the pole test.Current literature tends to show exacerbating effects of ri in PD  and a ca[H. suis . Thus, wH. suis/6-OHDA subgroups. A gastric H. suis infection thus seems to partially protect against 6-OHDA induced loss of dopaminergic input in the striatum. We further compared brain gliosis between the different subgroups by analyzing microglial and astrocyte activation in the striatum. While microglial activation was demonstrated to be specific for the 6-OHDA-injected striatum, astrocyte activation was demonstrated in both vehicle- and 6-OHDA-injected striata, but more pronounced in the 6-OHDA-injected striata. This suggests that the intrastriatal injection itself caused an activation of astrocytes, but not of microglia in the striatum, and that intrastriatal 6-OHDA injection clearly results in activation of both microglia and astrocytes in the injected striatum. Indeed, astroglial and microglial activation in the nigrostriatal pathway is described in PD patients [H. suis infection did not have an impact on the 6-OHDA-induced gliosis.In accordance with the motor function, the percentage loss of dopaminergic neuron fiber density in the injected versus non-injected striatum in the control/6-OHDA subgroups was larger compared to that of the patients  and has patients . HoweverH. suis infection, specifically through pathways related to oxidative stress and mitochondrial dysfunction. Oxidative stress is an important common pathway in neurodegeneration and is tightly interconnected with the pathological hallmarks of PD, namely dopaminergic cell death and aggregation of alpha-synuclein [In our study, we observed a neuroprotective effect of ynuclein ,47. Dopaynuclein ,57,58,59H. suis might, similar to H. pylori, evoke a local oxidative stress reaction whereby superoxide radicals are produced by the bacteria themselves, and ROS and inducible nitric oxide synthase (iNOS) production by gastric epithelial cells and immune cells is induced [H. suis infection might thus result in priming of the brain for exogenous oxidative stress, thereby increasing the chances of cell survival. induced ,65,66,67 induced . This tr induced . A gastrH. suis-infected mice, we demonstrated an upregulation of endogenous peroxidases which was significant for Sod2 and Cat. Although the expression of Cat in the brain is low in steady-state, post-mortem brain tissue of PD patients shows evidence of increased expression [Endogenous peroxidases are important antioxidant enzymes through their removal of ROS. In the brain of pression . Importapression .Nrf2 and its downstream effectors which are important regulators of cell defense mechanisms against oxidative stress. In steady-state conditions, NRF2 is quiescent in the cytosol. However, in the case of redox imbalance, NRF2 translocates to the nucleus and binds to the antioxidant response element (ARE) which is found in the promotor of several antioxidant response genes such as heme oxygenase 1 (Hmox1), NADPH oxidases (Nox1 and Nox2), thioredoxins , glutathione synthesizing enzymes such as glutathione-disulfide reductase (Gsr) and glutamate-cysteine ligase (Gclc and Gclm). Nrf2 has been implicated in key determinants of PD pathogenesis, namely mitochondrial impairment, oxidative stress and synucleinopathy. Multiple PD animal models have already shown the importance of the Nrf2 pathway with overexpression protecting against cell loss and alpha-synuclein aggregation, while Nrf2 loss exacerbates neurodegeneration [Nrf2 pathway was established in a human PD cohort, linking this activation to both alpha-synuclein pathology and the clinical disease progression [Nrf2-regulated genes [Nrf2 can influence PD pathology, which is replicated in our study in the H. suis-infected mice.In addition, we showed an upregulation of neration ,75,76,77gression . Post-moed genes ,79,80,81H. suis infection are in the long-term after dopaminergic cell loss. To conclude, the moment of H. suis infection could also be important, namely before or after the onset of disease, and could have different effects on disease progression or symptoms accordingly.Importantly, there are some limitations to the current study. The 6-OHDA mouse model induces acute and pronounced dopaminergic neurodegeneration which contrasts with the slowly progressive disease in humans. In addition, the importance of oxidative stress could be magnified in this mouse model due to the inherent oxidative mechanism of action of 6-OHDA. In our current experiments, we sampled one week after 6-OHDA injection. It could be interesting to see what the effects of H. suis infection protects, at least partially, against 6-OHDA-induced dopaminergic cell loss and motor function impairment in a unilateral intrastriatal 6-OHDA PD mouse model. As a potential explanation for this protective effect, we found H. suis-mediated changes in genes related to the reduction of oxidative stress. Specifically, brain tissue gene expression changes in the Nrf2 pathway and endogenous peroxidases were shown to occur in parallel with the observed neuroprotective effect of H. suis. This study adds to the accumulating evidence that antioxidant response pathways, with an emphasis on Nrf2, are promising targets for therapeutic interventions in PD and that these can be targeted through gut bacteria.In summary, our study unexpectedly shows that a gastric Five-week-old female C57BL/6JOlaHsd mice (n = 40) were purchased from Envigo  and housed in a conventional animal facility. The animals were kept in filtertop cages with a 12 h light/12 h dark cycle. They had free access to an autoclaved commercial diet  and water, and they were monitored daily. All animal experiments were approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University, Belgium (EC2016/21 and EC2017/17).H. suis isolate HS1 was originally obtained according to the method previously described [2, 10% CO2, 5% O2) at 37°C on biphasic Brucella culture plates  supplemented with 20% inactivated fetal calf serum , 5 mg/L amphotericin B , Campylobacter selective supplement , and Vitox supplement (Oxoid). Brucella broth (Oxoid) was added on top of the agar to obtain biphasic culture conditions. The pH of both agar and broth was adjusted to 5 by adding HCl to a final concentration of 0.05%. The bacteria were passaged at least twice to ensure reliable growth. After incubation, the bacteria were harvested and the number of viable (motile) bacteria/mL was microscopically determined using an improved Neubauer counting chamber (Sigma-Aldrich). The bacteria were diluted to a final concentration of 1 × 108 viable bacteria/mL.The porcine escribed . The bac8 viable H. suis HS1 bacteria/mL. The remaining 10 (short-term infection) or 9 (long-term infection) mice served as negative controls and received the growth medium of the bacteria ). One month post-infection (short-term infection) and 17 months post-infection (long-term infection), mice were euthanized for further analysis by the administration of an overdose of ketamine/xylazine followed by decapitation.At the age of 6 weeks, 10 (short-term infection) or 11 (long-term infection) mice were intragastrically inoculated with 300 µL of a stock solution containing 1 × 10One week prior to sampling, mice were placed one by one in random order in an induction chamber and anesthesia was induced with 2–2.5% isoflurane  in pure oxygen with a delivery rate of 0.9 L/min until loss of paw withdrawal reflex. After induction, mice were moved to a stereotactic apparatus  where general anesthesia was maintained with 1.5–1.8% isoflurane delivered in 100% oxygen. Anesthetic depth was assessed regularly throughout the entire procedure by monitoring respiratory rate and using the toe and tail pinch. To prevent hypothermia, mice were placed on a heated pad. The mice were fixated in the stereotactic apparatus, eye ointment was applied to prevent corneal drying, and the head was shaved and disinfected with an ethanol swab. The skull was dissected using a midline skin incision, and after identification of bregma and lambda as reference points, a hole in the skull was drilled at the injection site using the stereotactic apparatus. The freshly made 6-OHDA hydrochloride  solution (16 µg/µL 6-OHDA in 0.2% ascorbic acid) or the vehicle solution  was then automatically drawn into the syringe. The syringe was slowly directed to the injection coordinates relative to bregma , and 3 mm ventral to bregma) of the left striatum and 1 µL of either the 6-OHDA solution or the vehicle solution was subsequently injected over 10 min. After injection, the needle was kept in place for another 10 min, after which the syringe was slowly retracted. Bone wax  was applied to close the drill hole, the skin was sutured and both lidocaine  and iso-Betadine  were applied. Once recovered from anesthesia, mice were placed into their home cage whereby additional warmth was provided. Daily post-operative follow-up was performed by checking the behavior, body weight, and temperature of the animals.H. suis/vehicle, and (4) H. suis/6-OHDA, with the first term indicating the intragastric infection and the second term indicating the left intrastriatal injection.Eventually, both the short-term group (n = 20) and long-term group (n = 20) consisted of 4 subgroups: (1) control/vehicle, (2) control/6-OHDA, (3) Behavior and motor function tests were performed at 2 different timepoints, namely at baseline and post-injection. Tests at baseline took place after intragastric inoculation and just before intrastriatal injection, i.e., 7 days pre-injection for the short-term group and 1–2 days pre-injection for the long-term group. Post-injection tests took place 5–6 days post-injection and 1–2 days prior to sampling. Four different tests  were carried out to gain insights into motor function and exploratory behavior of the mice before and after left intrastriatal 6-OHDA injection. The motor function tests were adapted from Fleming et al.  and wereFor the traversal beam test, two clean mouse cages were inverted on a tabletop and a beam of 1 m length consisting of four sections with narrowing widths  was placed on top of the inverted mouse cages. The home cage of the mice was placed on its side at the end of the beam so that the narrowest end of the beam led right into the home cage. The mice were picked up by the base of their tails and placed at the wide end of the beam. The time the mice needed to traverse the beam was recorded. For training trials , no grid was placed on the beam. For the testing trials , a mesh grid that corresponded to each beam width was placed on top of the beam. The mesh grid had 1 cm squares and left a 1 cm space between the grid and the beam surface. The mean of the 3 best testing trials at each timepoint was used for analysis.For the pole test, a 50 cm long vertical pole of 1 cm in diameter was wrapped with tape  and placed in a cage. Mice were placed head-down on top of the pole and time to descend into the cage was assessed. Each mouse underwent three or four training trials (immediately starting from the top of the pole), and five testing trials at each timepoint. The mean of the 3 best testing trials at each timepoint was used for analysis.For the footprint analysis, the front and hind feet of the mice were coated with non-toxic paint of a different color. A small corridor was formed by placing two inverted mouse cages at the side of the corridor, with the home cage placed on its side at the end of this corridor. Each animal was then allowed to walk on absorbent white paper into this corridor to its home cage. After 1 training run without paint and one with paint, data were collected from 2 test runs. The stride length was then measured as the distance between the hind feet pawprints of consecutive steps. The mean stride length of the 2 test runs at each timepoint was used for analysis.For the cylinder test, mice were placed in an open-top, clear plexiglass cylinder of 20 cm high with a diameter of 13 cm for 5 min. Forelimb activity while rearing against the wall of the cylinder was assessed, whereby only placement of the whole palm on the cylinder wall (indicative for its use for body support) was counted. Contact with either the left or right paw, as well as with both paws simultaneously was registered.g for plasma collection. For serum preparation, blood was collected in Eppendorfs and stored overnight at 4 °C, centrifugated twice for 15 min at 1000× g, after which serum was collected and stored at −70 °C. Mice were then transcardially perfused with D-Phosphate Buffered Saline (PBS)/heparin (0.2% heparin). The stomach was isolated, cut open via the greater curvature and rinsed with sterile Hank’s Buffered Salt solution . Samples from the corpus and the antrum of the stomach were collected and stored in RNAlater . A longitudinal section starting from the forestomach and comprising the corpus and antrum of the stomach and part of the duodenum was taken and fixated overnight in 10% formalin at 4 °C and embedded in paraffin for H&E staining.After sedation, blood was collected by cardiac puncture and immediately transferred to EDTA-tubes  and centrifugated at 4 °C for 15 min at 300× For the brain sampling, striatum dissection was the priority to allow for dopaminergic cell death as an outcome parameter. To isolate the brain slice containing the injection site, a vertical cut was made at the rostral border of the hypothalamus and at 2 mm caudal from the frontal lobe. This brain slice containing the injection site was dissected and stored overnight in 10% formalin at 4 °C and embedded in paraffin. The brain part rostral of the brain slice  was snap frozen for subsequent gene expression analysis. In the long-term group, this forebrain part did not contain the olfactory bulbs.H. suis [H. suis standard was generated as previously described [TM RT-qPCR System with a C1000 Thermal Cycler . The Bio-Rad CFX Manager (version 1.6) software was used for the quantification of H. suis DNA in the tissue samples.The gastric samples that were stored in RNAlater  were homogenized  and RNA and DNA were separated using TriReagent RT  according to the manufacturer’s instructions. The number of colonizing bacteria per mg tissue was determined in the DNA samples using Real-Time (RT)-quantitative (q) Polymerase Chain Reaction (PCR) specific for the detection of H. suis . The H. escribed . Both stTM Total RNA Mini Kit , and total RNA from corpus and antrum of the stomach was further purified using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Purity of RNA was determined by measuring the ratio of absorbance at 260 and 280 nm with NanoDrop equipment . Next, cDNA was synthesized using the Sensifast cDNA Synthesis kit (GC Biotech) for the brain samples and the iScriptTM cDNA synthesis kit  for the gastric samples. RT-qPCR analysis of the brain samples was performed on the Light Cycler 480 system (Roche) using the 2xSensifast SYBR No-Rox kit . RT-qPCR analysis of stomach samples was carried out on a CFX384TM RT-qPCR System with a C1000 Thermal Cycler (Bio-Rad) using the 2xSensimix Sybr No-Rox kit (Bioline). Expression levels were normalized to the expression of reference genes as determined by the geNorm Housekeeping Gene Selection Software  [Cerebral samples were first homogenized  and RNA and DNA were separated using Trizol  according to the manufacturer’s instructions. Total RNA from cerebral tissue was further purified using the Aurumsamples) . The priex/λem = 485/520 nm using the FLUOstar Omega plate reader (BMG Labtech) and relative gastrointestinal permeability was calculated .The gastrointestinal permeability was determined as previously described . BrieflyThe levels of interleukin (IL-)1β, IL-6, the murine IL-8 homolog keratinocyte chemoattractant (KC), IL-10, and IL-17a in plasma (short-term infection group) and serum (long-term infection group) were measured using the Bio-Plex cytokine assay (Bio-Rad) according to the manufacturer’s instructions. To evaluate gastric inflammation, paraffin sections of 5 µm were cut, deparaffinized and rehydrated. Sections were then stained with Hematoxylin Gill III Prosan (Merck) and Eosin Yellow  according to the standardized protocols. The hematoxylin and eosin (H&E) staining was applied to determine the intensity of the overall gastritis, using the modified visual analog scale similar to the adapted Updated Sydney System as previously described .2O2 in methanol (5 min), respectively. Immunostaining of the brain sections was then performed as described before [To evaluate brain tissue, paraffin sections of 5 µm were cut, deparaffinized and rehydrated. To allow for comparison between the different subgroups, similar regions in the striatum were stained for each antibody, as depicted in d before  using th2O2 in methanol for 10 min at room temperature followed by washing with PBS. Samples were then blocked with 5% goat serum in antibody diluent  for 30 min at room temperature, followed by overnight incubation at 4 °C with either primary anti-GFAP antibody  diluted in block buffer or with primary polyclonal rabbit anti-TH antibody . The next day, slides were rinsed with PBS, and slides for GFAP staining were then incubated with biotinylated goat anti-rabbit secondary antibody  for one hour at room temperature, once again rinsed with PBS and incubated for 30 min with VECTASTAIN® Elite ABC system . Slides for TH staining were then incubated with EnVision HRP anti-rabbit  for 1 h. After a final wash step with PBS, 3,3-diaminobenzidine  was added for 5 min. Finally, a counterstaining with hematoxylin was performed, the slides were dehydrated and mounted with xylene-based mounting medium. IBA1, GFAP, and TH stainings of the brain sections were visualized using Zeiss Axio Scan.Z1  followed by automated quantification using Fiji and QuPath software. Quantification of the percentage brown color was done via color thresholding with correction for the total amount of tissue. For glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH) immunostaining, heat-induced antigen retrieval was performed using citrate buffer , followed by washing in PBS. Endogenous peroxidase activity was blocked with 3% Hp < 0.05; ** 0.001 ≤ p < 0.01; *** 0.0001 ≤ p < 0.001; **** p < 0.0001.Statistical analysis was performed using GraphPad Prism. Data were analyzed by the two-way ANOVA test and are presented as means ± standard error of mean (SEM), unless stated otherwise. Significance levels are indicated on the graphs * 0.01 ≤


In patient requiring vasopressors, the radial artery pressure may underestimate the true central aortic pressure leading to unnecessary interventions. When using a femoral and a radial arterial line, this femoral-to-radial arterial pressure gradient (FR-APG) can be detected. Our main objective was to assess the accuracy of non-invasive blood pressure (NIBP) measures; specifically, measuring the gradient between the NIBP obtained at the brachial artery and the radial artery pressure and calculating the non-invasive brachial-to-radial arterial pressure gradient (NIBR-APG) to detect an FR-APG. The secondary objective was to assess the prevalence of the FR-APG in a targeted sample of critically ill patients.Adult patients in an intensive care unit requiring vasopressors and instrumented with a femoral and a radial artery line were selected. We recorded invasive radial and femoral arterial pressure, and brachial NIBP. Measurements were repeated each hour for 2 h. A significant FR-APG (our reference standard) was defined by either a mean arterial pressure (MAP) difference of more than 10 mmHg or a systolic arterial pressure (SAP) difference of more than 25 mmHg. The diagnostic accuracy of the NIBR-APG (our index test) to detect a significant FR-APG was estimated and the prevalence of an FR-APG was measured and correlated with the NIBR-APG.r2 = 0.36; p < 0.001) and MAP  NIBR-APG.Eighty-one patients aged 68 [IQR 58–75] years and an SAPS2 score of 35 (SD 7) were included from which 228 measurements were obtained. A significant FR-APG occurred in 15 patients with a prevalence of 18.5% [95%CI 10.8–28.7%]. Diabetes was significantly associated with a significant FR-APG. The use of a 11 mmHg difference in MAP between the NIBP at the brachial artery and the MAP of the radial artery led to a specificity of 92% , a sensitivity of 100%  and an AUC ROC of 0.93 [95%CI 0.81–0.99] to detect a significant FR-APG. SAP and MAP FR-APG correlated with SAP (NIBR-APG assessment can be used to detect a significant FR-APG which occur in one in every five critically ill patients requiring vasoactive agents. A significant radial-to-femoral pressure gradient occurs in nearly one-fifth of patients in ICU. Repeated non-invasive assessment of the radial-to-brachial pressure gradient was significantly correlated with the invasive radio-femoral arterial pressure gradient. Radial arterial pressure monitoring is a common practice in intensive care units (ICU) . HoweverRisk factors in detecting this abnormal arterial peripheral-to-central gradient have been identified during cardiac surgery, but the diagnostic accuracy of such an approach is low , 10. ThiThese observations raise the question about the possibility to detect such a gradient in clinical practice, and eventually identify the population which need an invasive assessment, beyond the sole radial measurement. Several studies have shown that femoral and aortic arterial pressure were equivalents , 17, butThis prospective study was conducted in Montreal, Canada in three academic medical centers: over a 22-month period. The local Institutional Review Board for human subjects approved the study protocol (IRB#: MP-33-2016-1964) and informed consent was obtained before inclusion. We reported our study, regarding the methodological limits and the risk of bias, according to the STARD statement . Patient2), cerebral near-infrared spectroscopy (NIRS), fluid balance at the time of the measurement if available and central body temperature values were noted. The Sequential Organ Failure Assessment Score  had a significant SAP FR-APG and 10 (12%) [95%CI 6–22] had a significant MAP FR-APG. At T2, 10 patients (13%) [95%CI 7–23] had both a significant SAP FR-APG and MAP FR-APG. At T3, 7 patients 10% [95%CI 7–24] had a significant SAP FR-APG and 3 patients (4%) [95%CI 1–12] had a significant MAP FR-APG.The main finding of our study is that 15 patients among 81 patients (18.5% [95%CI 11–29%]) had a significant FR-APG in the ICU. Patients with abnormal FR-APG were more frequently diabetes, with higher lactate and received higher doses of norepinephrine. We also found that the average measurements of the two arms every hour, three times, of the NIBR-APG was significantly correlated with the FR-APG. Such measurement can diagnose a significant FR-APG with an AUC ROC of 0.93 [95%CI 0.81–0.99]. The best threshold was a 11 mmHg difference between the MAP of the brachial NIBP and the MAP of the radial artery pressure, with a specificity of 92%  and a sensitivity of 100% .The prevalence of a significant gradient on our selected group of patients was lower than what has been described during cardiac surgery, but is concordant with another similar ICU study . In Kim There is a discrepancy in the different studies in the identification of risk factors of a significant gradient. As previously described in the ICU, significant gradient is associated with higher doses of vasopressors , 39, 40.Kanazawa et al. studied different site of measurement along the radial, brachial and the subclavian artery. They found a strong correlation between the distance from the aorta to the site of measurement and the pressure drop from the aorta to the site of measurement . Those fThis study has several limitations. First, the association of FR-APG with diabetes has not been reported previously but our number of patients with a significant FR-APG is too small to identify more than one factor using logistic regression. This is, however, the largest reported study with radial and femoral artery measurements in the ICU after Mignini who included 55 patients  and the There are numerous advantages of continuous invasive arterial pressure measurement even through the radial artery which include blood gases measurements. If a discrepancy occurs and persist between non-invasive measurements and the radial artery, inserting a femoral arterial line would be suggested. Finally, an indication bias might be present. The use of both radial and femoral arterial pressure monitoring might have been decided for patients at risk of developing such gradients. However, in our institution, the use of both monitoring site is routinely used in up to 70% of patients in cardiac surgical patients , 43.Significant FR-APG can occur in nearly 1 out of 5 ICU patients. Such can affect significantly the interpretation of the hemodynamic condition of our patient with a risk of fluid or vasopressors overuse. No clear risk factors were linked strongly enough to enable the clinician to rule out the presence of that condition on an epidemiological approach. The NIBP measurement of brachial arterial using a MAP threshold of 11 mmHg can identify those patients and may avoid the use of an invasive femoral catheter. Persistence of a gradient despite modification of therapeutic strategy could warrant more invasive diagnostic strategy. Validation of this hypothesis in a larger cohort of patients should be performed.


Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of  How double strand breaks (DSBs) are repaired within the plant 45S rDNA repeats is unclear. Here, the authors show that Cas9-mediated DSBs in 45S rDNA are mainly repaired by cNHEJ and describe CRISPR-Kill as a tool for organ-specific cell elimination by targeting functional repetitive DNA in Arabidopsis. In plants, CRISPR/Cas systems are now widely used for genome editing in basic science as well as for trait improvement in breeding5. Beside that, CRISPR/Cas can also be used to study DSB repair itself. Multiple studies on DSB repair in unique genomic sequences have been performed but very little is known on how repeats are repaired within repetitive sequences6.Since its first description as programmable nucleaseArabidopsis thaliana are arranged in up to 750 10-kb-long tandem repeats per haploid genome8. Each repeat consists of the 18S, 5.8S, and 25S rRNA genes, separated by two conserved internal transcribed spacers (ITS1 and ITS2) and a variable intergenic spacer (IGS).The ribosomal RNA genes (rDNA) of 9. In somatic plant cells, the error-prone NHEJ pathway is the prevalent repair mechanism, which can be subdivided into the KU-heterodimer- and LIG4-dependent classical NHEJ (cNHEJ) pathway and the POLQ-dependent microhomology-mediated end-joining (MMEJ) pathway. Whereas cNHEJ mediates the direct ligation of the broken ends, often resulting in smaller insertions and deletions, MMEJ utilizes microhomologies (MH) close to the break sites, resulting in a loss of sequence between the MH11. Repair via HR requires a template and, in the case of tandem repeats, might result in repeat loss. Recently, it was shown that HR is downregulated within the 45S rDNA during meiosis, and that DSBs within this region are repaired via classical NHEJ (cNHEJ)12. However, it has been shown that HR plays a major role in the somatic repeat loss of 45S rDNA in the caf-1 mutant background in Arabidopsis13. To clarify the situation, the initial goal of this study was to directly induce DSBs within the 45S rDNA repeats by Cas9 to define by which pathway they are preferentially repaired. Here, we show that Cas9-mediated DSBs in 45S rDNA are mainly repaired by cNHEJ and furthermore describe a system for the controlled induction of cell death by targeting functional repetitive DNA.DSBs can be repaired by two pathways: non-homologous end-joining (NHEJ) or homologous recombination (HR)Staphylococcus aureus Cas9 (SaCas9)14. The target site was selected in the variable IGS region, so that only a subset of repeats was cleaved  which induces a unique DSB. Col-0 plants were transformed with the expression vector, Ubi-SaCas9-IGS, as well as the control vector. After two weeks of growth, 40 transgenic plants per line were selected and pooled for extraction of genomic DNA. Subsequently, the 45S rDNA target site was amplified and subjected to next-generation sequencing (NGS) to determine the repair pattern. The deep sequencing analysis revealed that the majority of mutations at the break site were deletions, most of them smaller than 10 bp, and that the repair occurred without MH  was used as a control, which showed an editing efficiency of 60% , was used for DSB induction16. This difference might have been caused by the fact that SaCas9 is superior in DSB induction in comparison to SpCas9 in Arabidopsis17. Our results clearly demonstrate that targeting the conserved part of the 45S rDNA with a highly efficient nuclease is lethal for the transformed cells  regions as target sites, which are conserved between all repeats. Two gRNAs were designed for ITS1 (Ubi-SaCas9-ITS1-1 and Ubi-SaCas9-ITS1-2) and one guide for ITS2 (Ubi-SaCas9-ITS2-1) . AP1 is a key player for flower meristem identity. It is highly expressed at the early stages of flower development and later gets restricted to specific tissue where it is required for sepal and petal development18 , characterized by a disturbed floral architecture. To determine whether there is a correlation between the strength of the phenotype and the amount of induced DSBs in the respective line, we isolated floral tissue and performed deep sequencing analysis of the ITS2 target site. In total, two ADH1 control lines (#81 and #82), as well as ten ITS-2-1 lines were analyzed for InDels. The latter ones were subdivided based on their phenotype in ‘no phenotype’ (#5 and #3), ‘intermediate phenotype’ , and ‘strong phenotype’ (#26 to #30)   Fig. . Transfoen) Fig. . In conten) Fig. . The pheion Fig. . InteresA. thaliana, the pericycle cells form lateral roots  promoter, which is exclusively expressed in the pericycle22, we transformed Col-0 plants with XPP-SaCas9-ITS2-1, XPP-SaCas9-Cen constructs, and pXPP-SaCas9-ADH1 as a control . Both systems have been used in plants before to eliminate certain flower tissues, i.e., reproductive organs. In tobacco and Arabidopsis, the barnase system could be applied to induce female sterility, by targeting the stigmatic secretory zone of pistils, or male sterility, by targeting the mature stamen, respectively26. Similarly, using DTA in combination with the promoter of the floral homeotic protein AP3, expressed in petal and stamen tissue, the respective tissue could be eliminated and, thus, male sterility could be induced27. Next to toxins, also the UV laser ablation technique has been used successfully for cell elimination, i.e., in root endodermis cells to analyze wound response and regeneration processes29.Taken together, we were able to demonstrate that SaCas9-mediated induction of multiple DSBs in 45S rDNA and centromere repeats can be harnessed to trigger cell death in a cell-autonomous way, resulting in the highly efficient elimination of selected plant cell types. A broad spectrum of phenotypic outcomes was generated, indicating that this system might be a valuable tool to accomplish various stages of cell type or organ elimination. The CRISPR-Kill approach differs massively from others, which conditionally knock out genes in specific cell types to prevent pleiotropic effects or lethality31. Transferring this to plants, CRISPR-Kill-based chromosome elimination might be an attractive application in plant hybrids, especially if coupled with chromosome restructuring33.The major advantage of the CRISPR-Kill system is the broad range of applications. Besides tissue elimination, it can be applied for purposes not aiming for cell elimination but rather genome engineering. In contrast to the classical methods that eliminate cells in a rampant manner, the CRISPR-Kill system targets specific genomic sites and, thus, can be programmed to cleave individual chromosomes. So far, it has only been shown in mammals that individual chromosomes can be eliminated through the induction of multiple DSBs34.The CRISPR-Kill system is easy to adapt for use in other plant species, or even other multicellular eukaryotes, such as animals or fungi, due to the high conservation of the target regions. It will be interesting to combine it with an inducible and two-component system to control Cas9 expression, so that CRISPR-Kill can be controlled both temporally and spatially35. Using the CRISPR-Kill system, the change of secondary metabolite composition within a plant and also the elimination or even production of new specific metabolites might become achievable. Thus, food production as well as pharmaceutical applications could benefit from this technology.CRISPR-Kill could also be an attractive tool for applications in synthetic biology. Cell-type specificity is a common characteristic of multiple biochemical pathways in plants, especially in secondary metabolism. Metabolites important for plant defense are often produced in specific cell types to avoid the peril of autotoxicity and to maximize the defense functionhttps://www.thermofisher.com) pDe-Sa-Cas9 and pEn-Sa-Chimera plasmids14. The kanamycin resistance cassette was exchanged with gentamycin using the restriction enzymes PmeI and SbfI. The target sequences for break induction were ligated with pEn-Sa-Chimera as oligonucleotides, using AS1-AS12  and later amplified using the Primer combination AS15 and AS16 , the PcUbi4-2 of Ubi-SaCas9-ADH1 and Ubi-SaCas9-ITS2-1 and Ubi-SaCas9-Cen were exchanged with the AP1 and XPP promoter to generate the tissue-specific constructs. For alignment and analysis of Sanger sequencing data, the software ApE was used. All constructs are available on request.DNA constructs were generated using the Gateway®-compatible . In addition, the T-DNA insertion lines ku70-1 36, lig4-5 37, and rad54-1 15 were obtained from the SALK collection38. Plants were cultivated in the greenhouse on soil  with 16 h light  and 8 h darkness at 22 °C. When sterile conditions were required, plants were cultured in a CLU-36L4 plant culture chamber  with stable conditions of 16 h light at 22 °C and 8 h dark at 20 °C on germination media . Seeds were surface-sterilized using 4% sodium hypochlorite and stratified overnight at 4 °C. For Agrobacterium-mediated plant transformation, floral-dipping was performed.Experiments were performed in the 39) . Parameters were set for minimum frequency (n) = 1 and comparison range R = 200, no wild-type marker (r). Afterward, the data were analyzed using R Studio and Excel.For next-generation sequencing analyses (NGS) of the ADH1 locus, the genomic DNA of a pool of 40 primary transformants was extracted. To analyze repair patterns and SaCas9 efficiency, primers with 6 bp overhangs , 0.5 μL of forward and reverse primer . To analyze phenotypical differences in the root, plants were first selected for transgene integration on selection medium containing PPT for 13 days. In total, 108 control plants of each line were transferred from selection medium to germination medium. After another 7 days, the number of lateral roots was determined. In addition, relative lateral root length, in relation to the main root length, was determined by the Image J add-on SmartRoot40. To analyze the effects of CRISPR-Kill in roots in the T2 generation, T1 lines were propagated to T2 and selected for heterozygous T-DNA integration on selection medium containing PPT. In total, three plants each out of 30 T2 lines were analyzed as described above.To analyze phenotypical differences in the floral tissue, plants were grown in the greenhouse for 4 weeks. In total, 19 control plants, 133 pAP1-SaCas9-ITS2-1, and 40 pAP1-SaCas9-Cen lines were screened for phenotypical differences and sorted into different categories, dependening on the extent of these differences. Analysis was performed using a binocular microscope and pictures were taken with VisiCam 5 plus using the software IS Visicam Image analyser (VWR, t-test with unequal variance. For root number and relative lateral root length, a one-way ANOVA was performed: ns P > 0.05, *P ≤ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Differences in survival rates were determined by applying a two-sided, two-sample Further information on research design is available in the Supplementary InformationReporting Summary


The relative importance of damage-associated and insect associated-factors in this induction of cotton volatile emissions is not yet fully clear. We evaluated how cotton plants respond to mechanical damage and to the application of the oral secretion from the generalist lepidopteran pest Spodoptera exigua, by measuring the local and systemic emissions of volatile compounds from their leaves. Our results confirm that cotton plants respond to damage-associated molecular patterns (DAMPs) as well as to herbivore-associated molecular patterns (HAMPs) present in the caterpillars’ oral secretion. Interestingly, a stronger response was observed for cotton plants that were treated with oral secretion from cotton-fed caterpillars than those fed on maize. We tested the possibility that volicitin, a common fatty acid-derived elicitor in caterpillar regurgitant plays a role in this difference. Volicitin and volicitin-like compounds were detected in equal amounts in the oral secretion of S. exigua fed on either cotton or maize leaves. We conclude that other elicitors must be involved. The identification of these eliciting cues is expected to contribute to the development of novel strategies to enhance the resistance of cotton plants to insect pests.In response to herbivore attack, plants release large amounts of volatiles that can serve as attractants for the natural enemies of the attacking herbivores. Such responses are typically triggered by damage- and insect-associated factors. Cotton plants are somewhat peculiar because they release specific blends of volatiles in two waves in response to caterpillar attack. They first emit constitutively stored volatile compounds, and after about 24 h a second wave that includes various  During the millions of years of interaction with herbivorous insects, plants have evolved a multitude of defense strategies. These include constitutive defenses as well as induced defenses, which are produced only upon herbivore attack . Among tPieris brassicae fed on cabbage, which triggers the release of volatiles that attract the parasitic wasp Cotesia glomerata  is a plant of great economic importance. It is known to have direct defenses such as gossypol, a terpenoid with insecticidal properties, and indirect defenses such as extra-floral nectar and HIPVs  were obtained from CIRAD . Seeds were soaked in tap water and covered with aluminum foil for 24 h at 24°C. Subsequently, they were kept in plastic boxes with a 4-cm layer of moist vermiculite until germination. Around 4 days after germination, the seedlings were transplanted to individual plastic pots filled with plant substrate soil . The plants were grown under greenhouse conditions  and watered every 2 days. All the plants used in the experiments had either two or four fully developed leaves. Maize seeds  were purchased from DSP Delley seeds and plants genetics Ltd.,  and they were germinated in individual plastic pots filled with substrate soil  and kept under greenhouse conditions (as above). The plants used in the experiments were 10 days old.Cultivated cotton seeds  eggs were purchased from Entocare . The caterpillars were reared on wheat-germ based artificial diet  under laboratory conditions . Regurgitant (R) was collected according to S. exigua fed on cotton plants, we performed a series of experiments to measure local and systemic volatile emissions over time. First, we carried out an experiment with cotton plants that had two developed leaves and were induced by simulating caterpillar damage and applying regurgitant to the wounds (n = 4–5) that were either mechanically damaged (MD), were MD and with application of regurgitant from S. exigua fed on cotton plants (MD + CR) and were mechanically damaged and with application of regurgitant from S. exigua fed on maize plants (MD + MR). Control plants were not damaged. Mechanical damage was inflicted by scratching one leaf (~2 cm2) with a pair of serrated forceps. Three sites on each side of the leaf were damaged in this way, and 10 μl of regurgitant  was immediately applied directly onto the wounded sites . However, this time the two lower leaves (1st and 2nd leaves) were treated, and after the final induction were removed, and the volatiles were collected only from the untreated 3rd and 4th leaves (n = 6) were induced twice a day (morning and evening) for 2 consecutive days. The plants were induced by MD, mechanical damage with regurgitant from S. exigua fed on cotton plants (MD + CR), and mechanical damaged with regurgitant from S. exigua fed on maize plants (MD + MR). Control plants were not damaged, but the two lower leaves were also removed. On the 3rd day, after 2 days of being induced by simulated herbivory, the plants received the last induction treatment at 8 am. The damaged leaves (1st and 2nd leaves) were removed 2 h later at 10 am, and the collection of systemic HIPVs was immediately performed.To evaluate the importance of HAMPs in the systemic response of HIPVs in cotton plants, we collected emitted volatiles from plants induced with h leaves –E. CottoS. exigua fed on cotton plants (CR), cotton plants incubated in regurgitant from S. exigua fed on maize plants (MR), and maize plants incubated in regurgitant from S. exigua fed on maize (MR). Based on the previous study, maize plants are known to respond to this form of induction, and  coupled with a mass spectrometer (Agilent 5977B GC/MSD) on TIC mode. About 2 μl of sample were injected in pulsed splitless mode onto an Agilent HP-5MS column (30 m length x 0.25 mm diameter and 0.25 μm film thickness). The temperature program was as follows: kept at 40°C for 3 min, increased to 100°C at a rate of 8°C minS. exigua fed on cotton plants produce elicitors, we measured relative amounts of volicitin and volicitin-like compounds content in regurgitant of S. exigua caterpillars fed on either cotton or maize plants. FACs, specially volicitin N-(17-hydroxylinolenoyl)-L-glutamine were chosen based on previous results from S. exigua fed on maize plants produce this elicitor, which is responsible for inducing HIPV emission in maize plants. To this end, the regurgitant was collected as previously described, each sample (n = 5) corresponded to 10 μl of regurgitant collected from two caterpillars. For the extraction, 1 ml of MeOH:H2O 50% (50:50) was added to each sample, which was then vortexed and centrifuged at 10,000 rpm and at 4°C for 15 min. The supernatant was collected and used for volicitin analysis.In order to check whether m/z 145.0615 corresponding to a glutamine moiety). Peaks corresponding to volicitin were volicitins were automatically integrated using TargetLynx XS with a 0.1 min chromatographic window centered on the retention time of each compound and a 0.02 Da mass window centered on the (M-H)− ion.The analysis of volicitin was performed by UHPLC MS instrument (Waters) made up with using ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry. Specifically, an Acquity UPLC (Waters) coupled to a Synapt G2 high-resolution mass spectrometer. QTOF was employed. The column used for separation was Acquitting UPLC BEH C18 1.7 μm, 2.1 × 50 mm (Waters). The temperature was maintained at 25°C. Two eluants were used: water and 0.05% of formic acid (eluant A) and acetonitrile and 0.05% of formic acid (eluant B). A linear gradient from 10 to 100% B in 7 min was applied. The injection volume was 2.5 μl. The mass spectrometer source was operated in electrospray negative ionization and data were acquired in data-independent acquisition (DIA) mode . Exact mass measurements (<2 ppm) were ensured by infusing a 500 ng/ml solution of leucine-enkephalin at 15 μl/min through the Lockspray probe. For data acquisition and processing, we used the software Masslynx v.4.1 (Waters). Volicitin and related molecules were identified based on the determination of the most probable molecular formula as well as fragmentation pattern  were used to compare significant differences among treatments. An orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical clustering heatmap were carried using MetaboAnalyst 5.0 -4,8–dimethyl–1,3,7-nonatriene (DMNT), -4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), and indole were emitted only after 48 h of elicitation -β-farnesene were emitted mostly by plants treated with the regurgitant of cotton-fed caterpillars, whereas the monoterpene α-pinene were emitted by plants treated with both types of regurgitant, but in higher amounts by plants treated with the regurgitant of cotton-fed caterpillars : 18:3-OH-glutamine (volicitin), 18:2-OH-glutamine, 16:1-OH-glutamine, 18:01-OH-glutamine, 18:03-glutamine, and 18:2-glutamine. Interestingly, all the six FACs found in the regurgitant showed similar levels regardless of the plant food source that the insect fed on . One such receptor was recently identified inceptin -β-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and (E)-β-farnesene. These truly inducible volatiles are also systemically released from non-attacked leaves of cotton plants -4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), and indole in the systemic response. Indeed, S. exigua regurgitant solution did not result in any induction of HIPV. This is in sharp contrast to maize plants that respond strongly to the incubation in regurgitant solution of S. exigua with the emission of typical maize HIPVs (The overall blend of HIPV emitted by cotton plants treated with terpenes , 4. Thesterpenes . Previoule delay . In accon plants . The samze HIPVs , as we cze HIPVs . Importaze HIPVs . Testingze HIPVs .We draw two primary conclusions from our work investigating the factors involved in caterpillar induced cotton volatiles. Firstly, mechanical damage alone is not sufficient to induce a full response and elicitors present in caterpillar oral secretions enhance the response. Secondly, the difference in emissions from plants treated with cotton- and maize-derived secretions may imply that unidentified cotton DAMPs, help the plants to recognize self-damage. Cotton plants have been shown to particularly amenable to priming with inducible volatiles; they can become considerably resistant to insect attack if they have been exposed to volatiles from attacked plants . TherefoThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TT and CA designed the study. CA and GB collected data and analyzed and interpreted the data. GG developed the method to analyze volicitin. CA and GA wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Neurofibromatosis type 1 (NF-1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. It is characterized by multiple café-au-lait macules, cutaneous neurofibromas, optic glioma, Lisch nodules, and axillary and inguinal freckling. The aim of this study was to investigate NF1 mutations in two Chinese families with NF-1 who presented with early-onset hypertension, and to determine the prevalence of hypertension associated with NF-1 to better understand this complication.Methods: Whole-exome sequencing was performed for the probands with NF-1 from two unrelated families. Possible pathogenic mutation was predicted by bioinformatic tools. Sanger sequencing was used to confirm candidate variants in all available individuals for familial co-segregation analysis. We also performed a systematic literature review of studies that reported the prevalence of hypertension in patients with NF-1.Results: In family 1, a recurrent mutation c.6789_6792delTTAC in NF1 was identified in the proband but in no other family members, indicating that this is a de novo mutation. In family 2, a novel mutation c.6934_6936delGCAinsTGCT in NF1 was detected in the proband and two other family members, which co-segregated with the disease phenotype within the family. Both mutations were predicted to be pathogenic by bioinformatic analysis. We found hypertension was a relatively common complication of NF-1, with a prevalence range of 6.1–23.4%. Ambulatory blood pressure monitoring is a stable method for detecting initial alterations of the blood pressure pattern, particularly for pre-hypertension.Conclusions: We identified one recurrent (c.6789_6792delTTAC) and one novel frame-shift mutation (c.6934_6936delGCAinsTGCT) in two unrelated families with NF-1 using whole-exome sequencing. In consideration of phenotypic heterogeneity in NF-1, genetic testing is a robust tool which helps early and accurate diagnosis. Because hypertension is not a rare complication of NF-1, routine screening for hypertension in patients with NF-1, especially children and adolescents, is important to avoid serious cardiovascular events. NF1), a tumor suppressor gene mapping to 17q11.2, and spans 60 exons with an approximate size of 350 kb. NF1 encodes neurofibromin, which belongs to the Ras-guanosine triphosphatase (RAS-GTPase) activating protein family. Loss-of-function mutations in NF1 lead to intracellular neurofibromin protein deficiency, followed by over-activation of RAS signaling to the downstream pathways, which consequently leads to the development of phenotypes characterized by pigmentary lesions and predispositions to neoplastic disorders , also known as von Recklinghausen disease, is a progressive autosomal dominant disorder. NF-1 is one of the most common hereditary diseases with an estimated prevalence of 1 in 2,500 to 1 in 3,000 persons worldwide , no matter whether it is the essential form or secondary to renal or aortic vasculopathy, or pheochromocytoma . HyperteNF1 variant c.6789_6792delTTAC and one novel heterozygous mutation c.6934_6936delGCAinsTGCT in families 1 and 2, respectively. We also summarize the data from previous clinical studies into the hypertension associated with NF-1 to better understand this complication.Here, we investigated two unrelated Chinese families with NF-1 characterized by early-onset hypertension by whole-exome sequencing and Sanger sequencing and identified one recurrent Two unrelated families, comprising four patients with NF-1 and three unaffected individuals, were recruited for this study. The pedigree chart was shown in This study complied with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Fuwai Hospital. Informed consent forms were obtained in advance from all the recruited participants.http://broadinstitute.github.io/picard/). Subsequently, insertions/deletions and single nucleotide polymorphisms were identified using GATK (http://www.broadinstitute.org/gsa/wiki/index.php/Home_Page) and SOAPsnp (http://soap.genomics.org.cn/soapsnp.html). Variants were filtered in multiple databases, including the 1,000 Genomes Project database (http://browser.1000genomes.org/), dbSNP (https://www.ncbi.nlm.nih.gov/snp/), the Human Gene Mutation Database , and Exome Aggregation Consortium (http://exac.broadinstitute.org/). Additional in silico analyses were performed using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and MutationTaster (http://www.mutationtaster.org/) . The pathogenicity of potential mutations was evaluated according to the American College of Medical Genetics and Genomics guidelines for the interpretation of sequence variants that were published in 2015 . Genomic DNA was extracted from peripheral blood leukocytes from all the participants using a QIA Amp DNA Blood Mini kit  in accordance with the manufacturer's protocol. The concentration and quality of each DNA samples were evaluated by Thermal Nanodrop 2000 and agarose gel electrophoresis, respectively. To generate the DNA libraries, genomic DNA fragmentation, end repair, adapter ligation, and PCR enrichment were performed according to Illumina protocols. A GenCap exome capture kit (MyGenostics GenCap Enrichment technologies) was used for exome capture. DNA sequencing was performed on the Illumina HiSeq X Ten system. After removing Low-quality reads and adaptor sequences, the Burrows-Wheeler Aligner tool was used to align the clean reads to the human reference genome . Duplicate reads were removed using the Picard software (er.org/) . The neuSanger sequencing was used to confirm the candidate variants detected by the whole-exome sequencing within the two families. Co-segregation analyses were performed in relevant available individuals. The designed primer sequences are listed in We performed literature research using “neurofibromatosis type 1,” “von Recklinghausen disease,” “neurofibromatosis-1,” “hypertension,” “elevated blood pressure,” and “blood pressure” in the PubMed and Embase databases with no year limit. Publications that met the following predetermined criteria were excluded: (1) articles were not in English; (2) article types were book section, or review; (3) articles were related to pulmonary, gestational, intracranial, or intraocular hypertension; (4) animal models or fundamental experiments were involved. Only articles with subjects who were general patients with NF-1 were included in the review. Variables including author, years of publication, number of patients with NF-1, method of blood pressure measurement, blood pressure results, secondary causes of hypertension, and complications of NF-1 were extracted from the selected articles by two authors independently.Patient 1 was a 26-year-old man (proband II-1 in family 1) who was admitted to the Fuwai Hospital with suspected NF-1 and early-onset hypertension. When he was 6 years old, a mass with a diameter of 7 mm was found on his left palm, which was later confirmed as neurofibroma by pathological biopsy after resection . Gradually, multiple CALM and cutaneous neurofibromas appeared, distributing mainly on his abdomen and anterior and posterior thorax . When heFour individuals in family 2 participated in the study. The proband was a 14-year-old adolescent boy (III-1 in family 2) referred to hospital for early-onset hypertension with severe headache. Physical examination found mental retardation , multiple CALM on his neck and trunk, and axillary freckling . Head CTNF1 (NM_000267) were supposed as candidate causative variants associated with the phenotypes of probands II-1 (family 1) and III-1 (family 2), respectively. Both variants were predicted to be pathogenic by MutationTaster and have not been reported in the 1,000 Genome Project, ExAC_ALL, or ExAC_EAS databases  and another two family members , which co-segregated with the disease presentation in this family.The whole-exome sequencing of the samples from the two probands yielded approximate 17 GB and 11 GB raw data with more than 99% coverage of the target region. The average sequencing depths were 213 and 123 in proband II-1 (family 1) and III-1 (family 2), respectively, with more than 98% of the bases read at 10 × coverage and 97% bases read at 20 × coverage. Two heterozygous frame-shift mutations, c.6789_6792delTTAC and c.6934_6936delGCAinsTGCT in atabases . The resatabases . Moreoveatabases . Four NFThrough the literature search, we retrieved a total of 363 articles after excluding duplications; the selection flowchart is shown in NF1 among four affected individuals from two unrelated Chinese families using whole-exome sequencing. One mutation (c.6789_6792delTTAC) is a de novo mutation that has been reported previously; the other mutation (c.6934_6936delGCAinsTGCT) is reported here for the first time, to our knowledge, with evident pathogenicity predicted by the bioinformatic analysis and family co-segregation. Furthermore, by conducting a systematic literature review on patients with NF-1 associated with hypertension, we found that hypertension was a relatively common complication of NF-1, with a prevalence of 6.1–23.4%.In this study, we identified two frame-shift mutations in NF1, which encodes neurofibromin protein. Neurofibromin functions as a RAS-GTPase activating protein that accelerates the conversion of the active form RAS-GTP to the inactive form RAS-GDP  of family 1 , learning difficulties, and lacking forms of neurofibromas , the National Key Research and Development Program of China (2016YFC1300100).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tai-Chi is a popular indoor-based exercise that could induce several health-related benefits, but its benefits for middle-aged adults are unclear. Tai-Chi exercise can equally improve cardiorespiratory fitness, resting health rate, blood pressure and lean mass as a moderate-intensity running exercise. Running exercises appears to have further benefits in reducing the blood lipid level. The results from this study can provide insights into various types of exercises on cardiorespiratory fitness and biomarkers in the middle-aged population.2max, Vmax and Peak heart rate) was assessed at the baseline and the 12- and 24-week interventions. Results Compared to the no-exercise control group, both the Tai-Chi and running groups significantly decreased resting heart rate, diastolic blood pressure and cardiorespiratory fitness and increased lean mass across the training session (p < 0.05). Compared to the Tai-Chi group, the running group showed greater improvement in VO2max and Vmax (p < 0.05) and reduced triglyceride and low-density lipoprotein cholesterol (p < 0.05). Conclusion Both Tai-Chi and running exercise showed beneficial effects on cardiorespiratory fitness and enhanced health-related outcomes in middle-aged adults. Although Tai-Chi exercises were less effective in VO2max than running, Tai-Chi may be considered as a plausible alternative to running exercises that can be achieved in the indoor-based setting.This study examined the effectiveness of Tai-Chi and running exercises on cardiorespiratory fitness and biomarkers in sedentary middle-aged adults under 24 weeks of supervised training. Methods Thirty-six healthy middle-aged adults (55.6 ± 5.3 yr) were randomly assigned into Tai-Chi, running and control groups. During a 24-week training period, the Tai-Chi and running groups were asked to perform exercises for 60 min/day and 5 days/week, which were supervised by Tai-Chi and running instructors throughout. Resting heart rate, lean mass, blood pressure and blood lipids were measured, and cardiorespiratory fitness (VO Cardiorespiratory fitness refers to the functional capacity of the circulatory and respiratory systems to supply oxygen for the energy production needed during physical activity. It is considered to be an effective and sensitive predictor of cardiovascular disease and other chronic diseases ,2,3. A m2max). According to the American College of Sports Medicine (ACSM) recommendation, the exercise intensity is prescribed between 64% and 90% of the maximal heart rate (HRmax) to improve the myocardial adaptability [2max improvement than exercises with fewer exercise sessions. There are several exercise recommendations to improve cardiorespiratory fitness in the literature ,5. The otability  suggeste2max in sedentary older adults [To date, common aerobic exercises such as swimming, jogging, and running are recommended as they are easy to assess and also improve cardiorespiratory fitness. While Tai-Chi is a popular form of exercise in Asian countries, as well as worldwide ,9, littlr adults ,18. However, investigations have been predominantly focused on older adults, so the results may not be directly applied on middle-aged adults who have clear differences in physical condition and movement patterns. Physical activity research on the middle-aged population would be of great benefit for the quality of life and fitness in the older age population. Moreover, most of the studies comparing different types of exercises did not strictly control the frequency/duration and nutrient intake. It is questionable if Tai-Chi would be more beneficial on cardiorespiratory fitness than the commonest prescribed exercises, such as running, under the same amount of exercise (frequency/duration and nutrient intake) in the middle-aged population. Hence, the aim of this study was to compare the effects of Tai-Chi and running exercises on cardiorespiratory fitness and cardiometabolic biomarkers under 24-week supervised training. The results from this study can provide insights and application guidelines of exercises for the healthy, sedentary middle-aged population. 2, or they had coronary artery disease, anemia or other cardiovascular diseases. A total of 45 male middle-aged adults were recruited with an incentive of USD 350 for this 24-week supervised exercise study. The participants were randomly assigned into each of three exercise groups . There were nine participants who withdrew from this study due to sickness (n = 3) and unable to fit their personal schedule (n = 6). The others completed the entire 24-week exercise session. Eventually, a total of 36 participants completed the entire 24-week exercise sessions (120 days) for the Tai-Chi (n = 10), running (n = 14) and control (n = 12) groups. The demographic characteristics of the participants are shown in Healthy sedentary middle-aged male adults were recruited from a local community. All of them were reported to have fewer than 1000 steps each day and no regular exercise habits. An Actigraph accelerometer  on the right hip was used over consecutive days to confirm all participants had fewer than 1000 steps per day on average . They wemax was calculated by 207–0.7 * year for males [2max for individual participants at baseline, based on the Bruce protocol  for Tai-Chi, running and control group participants. The 50–60% of the VO2max level was also equivalent to about the running speed of 5.8–7.2km/h and step frequency of 150–158 step/min used for the running group.Participants were randomly assigned into Tai-Chi, running and control groups in this study. The HRor males . All paror males . All of max, specified in our pilot study. Participants acquired a full set of Tai-Chi techniques including breathing, balance, flexibility, concentration, calming, and stress-reduction techniques. During the early learning sessions, the instructors taught 4 movements per session, and this accumulated as sessions increased. All participants learned all movements of Chen-style Tai-Chi within the first four weeks. After that, the participants repeatedly performed the same Tai-Chi movements under the supervision of the same Tai-Chi instructor. Prior to each exercise session, participants were asked to perform a 20 min warm-up and activated the alarm of the heart rate monitor, which provided haptic vibration feedback on the wrist if the participant’s heart rate did not lie within the range of 66–74% HRmax  at any time during exercise. The instructors monitored the performance of each participant and confirmed that all participants had good compliance in their sessions. Participants in both Tai-Chi and running groups exercised outdoor between 7 and 9 am, 60 min per day and 5 days per week, for 24 weeks under supervised training. The total exercise duration was 7200 min/120 h (60 × 5 × 24 min) over the 24 weeks. The participants were required to wear the heart rate monitor watch  and Actigraph activity monitor during the entire experiment, including exercises, non-exercise activities and sleeping. Before the intervention, participants in the Tai-Chi group attended a 2 h class once per week for four weeks to learn the Chen style of Tai-Chi and each movement step was executed in about 60–65 steps/min, which has moderate intensity around 66–74% HRmax , which corresponds to a running speed of 5.8–7.2 km/h and a step frequency of 150–158 step/min. The range of heart rate and running speed/step frequency were predetermined with the 50–60% of the VO2max level before the start of the exercise intervention, which has previously been used in earlier studies [max  during the running sessions. Two experienced running instructors served as the pacemakers for the speed of 5.8 km/h and 7.2 km/h to ensure all participants were running with the same intensity throughout the 24-week experiment. Participants provided a training diary to record their running mileage, which was monitored, and they were reminded not to have a significant change in their non-exercise activities weekly by telephone calls. The training diary was collected after the final running session.Participants in the running group performed running exercise at 66–74% HR studies ,22. SincParticipants in the control group were asked to maintain their physical activity level and nutrient uptake as they acquired in the briefing and nutrient lectures . The parFor the Tai-Chi, running and control groups, the participants were invited to come back to our laboratory to complete the activity questionnaire (IPAQ) together with the reference of their weekly Actigraph data to determine the overall physical activity level at baseline (0 week), in the 12th week and in the 24th week. In all three groups, we used the Food Frequency Questionnaire (FFQ)  to measu+, SunTech Medical, Morrisville, NC, USA).At baseline, and after 12 and 24 weeks, all participants visited the laboratory. The participants were asked to complete the activity questionnaire (IPAQ) and foot frequency questionnaire (FFQ) as well as measure their body composition. Their cardiorespiratory fitness and biomarkers were assessed by the same exercise physiologist and registered nurse. After participants had sat quietly for 10 min, their resting heart rate (HR) was measured with a polar chest-belt monitor  and blood pressure, including systolic blood pressure (SBP) and diastolic blood pressure (DBP), was measured with an electronic sphygmomanometer . Plasma was isolated by centrifugation at 3500 rpm for 10 min and frozen at −80 °C within two hours of collection. Analyses were subsequently completed within three months of collection. Five metabolic indicators, including plasma triglycerides (TG), total cholesterol (TC), high and low-density lipoprotein cholesterols (HDL-C and LDL-C), and fasting blood glucose (FBG), were determined.2max and maximum aerobic speed (Vmax), which is in accordance with the Bruce protocol [max is determined as the maximum termination velocity when the participants finished their graded exercise tests. We asked the participants to give their RPE throughout the graded exercise test. The RPE at the 50–60% VO2max level was extracted to allow comparisons among the Tai-Chi, running and control groups, as this VO2max range was used to set the exercise intensity for our running and exercise groups. Participants were instructed to avoid any intake of medicine or alcohol and not to perform any vigorous physical activity for 12 h before the running test. During the graded exercise tests, peak heart rate (HRpeak) and blood pressure were monitored. The VO2max tests were analyzed breath-by-breath and by calculating the mean values from expired air at 30 s intervals using a metabolic measurement device .Graded exercise test design was used to assess the cardiorespiratory fitness, including VOprotocol . Vmax isAll statistical analyses were performed using SPSS 17.0. The data normality was examined with Shapiro–Wilk tests for each of the dependent variables and the findings indicated that the data in all variables were normally distributed, except for HDL, LDL and FBG. The Geisser’s epsilon adjustment was applied when the Mauchley test found a violation in the sphericity assumption. Levene’s test was also used to assess the equality of variances between different groups. Two-way (3 exercise × 3 time) factorial ANOVA with repeated measures was used to determine the interaction and main effects for each of the tested variables. When a statistically significant difference was observed, a Bonferroni post hoc comparison  was performed. The alpha was set at 0.05 for all analyses. p < 0.001) and self-reported physical activity , while the control group remained robust across the training time (p > 0.05). The group effect indicated a higher number of steps per day in Tai-Chi and running than the control (p < 0.001), while no significant group difference for the self-reported physical activity was found (p > 0.05).There was a significant interaction between group and time in average number of steps per day , while levels remained unchanged across training time in the control group (p > 0.05). For nutrient intakes, significant interactions were determined in total dietary carbohydrate, protein and fat intakes (p < 0.005). The simple main analysis indicated that the magnitude of the resting HR was decreased by 13.8 ± 2.3 b/min (p < 0.001) in the Tai-Chi group and −3.7 ± 1.5 b/min, (p = 0.021) in the running group after the 24-week intervention. The analysis also revealed that lean mass increased over the 24-week training in the running and Tai-Chi groups (p < 0.05), but not changed in the control group. There was a significant interaction between group and time in resting HR, body mass and lean mass (p = 0.006), but not in SBP (p > 0.05). After 24-week intervention, the Tai-Chi group  and running group  DBP was significantly lower than at baseline. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) are shown in p < 0.001) and LDL-C (p = 0.012), but not the others . The TG concentration decreased in the running group after 12 weeks of intervention , and this decrease continued until the end of 24 weeks . Although an increasing trend of LDL-C was found in the control group from baseline to 24 weeks (p < 0.05), there was a continual decline in the running group by −0.37 ± 0.18 mmol/L (p = 0.048) in 12-week and −0.46 ± 0.18 mmol/L (p = 0.020) at 24-week intervention. The blood test results  are shown in 2max (p < 0.001) and Vmax (p < 0.001). The simple main effect revealed that the maximal MET achieved in Bruce protocol was increased but PRE score was decreased across the training in the Tai-Chi and running groups (p < 0.05), but it remained similar across training times in the control group. The analysis also revealed that VO2max (mL/kg/min) increased in both exercise intervention groups, while in the control group, the VO2max remained unchanged throughout the 24-week study period, p < 0.001). The VO2max significantly increased after the 12-week intervention with Tai-Chi group  and the running group  and continued to increase until the 24-week intervention . The changes in VO2max in the running group were greater than in the Tai-Chi group after the 24-week intervention (p < 0.001).The interaction between group and time was found in maximal MET achieved in Bruce protocol, VOmax had increased by 0.8 ± 0.2 km/h (p = 0.001) and 1.4 ± 0.3 km/h (p < 0.001) in the Tai-Chi group and increased by 1.3 ± 0.2 km/h (p < 0.001) and 1.9 ± 0.3 km/h (p < 0.001) in the running group at 12 weeks and 24 weeks, respectively. There were significant differences in Vmax between the two exercise groups in the 12-week (p = 0.019), but not the 24-week, intervention (p > 0.05). Furthermore, HRpeak was found to increase from baseline by 14.8 ± 3.8 b/min in the Tai-Chi group and 9.5 ± 3.2 b/min in the running group after 24-week  and dietary intake for both the Tai-Chi and running groups. The total amount of exercise was based on the ACSM [2max obtained in the Bruce graded exercise at the start of the experiments. This intensity level is considered to be moderate, which is designed for our participants who had no regular exercise habits and less than 1000 steps per day. For the Tai-Chi and running groups, movement intensity was supervised by instructors and participants were alerted by the haptic vibration feedback from the heart rate monitor during each exercise session. This arrangement was used to determine the objective comparison between exercise interventions. Compared to baseline, both the Tai-Chi and running groups increased their number of steps per day at 12 weeks and 24 weeks, while the no-exercise control group had a similar number of steps per day across training time. This confirmed that our exercise intervention was effective in promoting physical activity.This study examined the effectiveness of Tai-Chi and running exercises on VOBoth the Tai-Chi and running groups showed significantly lower resting heart rates than the no-exercise control at 24 weeks, but no differences between groups were found at baseline and 12 weeks. The reduced resting heart rates in both exercise groups could be due to the improvement in cardiorespiratory capacity as well as body and blood composition  after the 24-week supervised training. While the Tai-Chi group appeared to have a higher resting heart rate than the other two groups, further investigation with larger sample sizes should be considered to reduce the impact of the individual subject differences before a viable conclusion can be made.2max of both intervention groups significantly increased over the intervention period; however, the increase in the running group was greater than in the Tai-Chi group after the 24-week intervention. The running exercise group appeared to induce more benefits related to cardiorespiratory fitness than the Tai-Chi group. One plausible explanation could be that running exercise mainly involves lower-limb bi-pedal movements, which is highly similar to the movement task used in cardiorespiratory tests . This would activate more relevant motor units due to the increased efficient motor task transfer [Tai-Chi and running have different movement characteristics and their effects on cardiac function, peripheral blood circulation, proprioception and body control may also differ. The VOtransfer . Anothertransfer ,23. Whiltransfer . 2max. Although the improvements in cardiorespiratory fitness in Tai-Chi were less than in running exercises after 24-week supervised training in our study, Tai-Chi might promote dynamic balance and thus prevent falls, as shown in previous studies [Tai-Chi is a relatively “easy to assess” approach to maintain physical performance. It is characterized as the movement of Yi and Qi, with Qi driven by Yi that is controlled by the mind. The movements of Tai-Chi are relatively slow and are controlled by the depth and frequency of respirations. Tai-Chi is suggested to improve muscular elasticity and breathing, which is regulated by several small muscles in the chest and abdominal wall. Since the movements of Tai-Chi are light and smooth, the stimulatory effects are relatively small and Tai-Chi results in steady improvements in VO studies . Moreove studies  and redu studies ,27. FurtFat burning and lipolysis are slowly processed by implementing exercises with low and moderate intensities. Our findings indicated significant decreases in LDL-C and TG in the running group but a subtle decrease in the Tai-Chi group after the 24-week training. This is in line with the previous running and Tai-Chi studies ,29. Tsao2max and lean mass as well as decrease body mass, BMI and resting diastolic blood pressure, which are considered as the risk factors for cardiovascular diseases. Together with the current findings, regular exercises at moderate intensity would improve diastolic velocity and allow full cardiac ejection [Interestingly, the results from this study indicated both Tai-Chi and running exercise effectively reduced blood pressure (DBP) and increased lean mass at the end of 24-week intervention. Tai-Chi was proved to reduce blood pressure and strengthen both the systolic and diastolic functions of the cardiovascular systems, as similarly suggested in other aerobic exercises ,31. Blooejection ,35,36.2max level. There is a dose–response relationship between exercise and cardiorespiratory fitness [2max increased from baseline in both groups at 12 weeks and this improvement continued until the 24-week intervention. However, running exercises showed larger improvement in VO2max than Tai-Chi exercises after the 24-week intervention. While cardiac output is the key limiting factor to increase VO2max, it is associated with exercise intensity. After the 24-week intervention, both Vmax and peak heart rate were also increased during the graded exercise test, indicating a higher graded exercise performance (higher MET achieved in Bruce protocol and lower RPE score at 50–60% VO2max) for both the Tai-Chi and running exercise groups. The combination of these biomarkers could help to prevent cardiac stroke, keep stroke volume stable, and allow maximal oxygen uptake during the increase in physical activities.Cardiorespiratory fitness is one of the predictors of cardiovascular disease and other chronic diseases ,2. Other fitness ,38,39. IWhen interpreting our results, some limitations should be considered. First, only middle-aged Chinese males were recruited in this study. The results may not be generalizable to female and Western participants. Gender differences may potentially show different physical activity habits, body compositions and metabolic parameters , and car2max in both exercise groups suggest that Tai-Chi can be used as an alternative exercise to running for improving physical health in sedentary middle-aged adults.Both Tai-Chi and running exercises with moderate intensity can effectively reduce resting heart rate, diastolic blood pressure and cardiorespiratory fitness in sedentary middle-aged adults after 24-week supervised training. Increased lean mass, reduced blood pressure, and improved blood composition and VO


The extraction efficiency of copper from the scrap Cu/ITO thin film was >99% with IL-I. Using XRD, crystal In2O3 was found on the regenerated ITO thin film which had a resistivity similar to that of unused ITO thin film. Using X-ray absorption near edge structural (XANES) spectroscopy, at least two paths for the extraction of copper from the Cu/ITO thin film into IL-I were identified. Path I: Copper is stripped from the scrap Cu/ITO thin film and then interacts with I3− in the IL-I to form nano CuI. The nano CuI further interacts with I−. Path II: Copper interacts with I3− on the surface of the Cu/ITO thin film to form nano CuI. The nano CuI is further stripped into the IL-I to interact with I−. During extraction, the nanoparticle size could be increased in the IL-I by conglomeration due to fewer coordinating anions and decrease in the viscosity of IL-I at high temperatures. Furthermore, nanoparticle growth was affected by [PF6]− of the IL-I determined via 31P NMR.Copper is coated on indium-tin-oxide (ITO) thin film to improve its electrical resistivity. In order to recycle the scrap Cu/ITO thin film, an ionic liquid (1-butyl-3-methyl imidazolium hexafluorophosphate ([C The resistivity and crystal phase of the regenerated ITO thin film was similar to that of unused ITO thin film [4mim][PF6], containing iodine/iodide (IL-I), was used to extract Cu from Cu/ITO. The main objective of the present study was to investigate the chemical structure of extracted copper from discarded Cu/ITO thin films in IL-I at 303–543 K by in situ X-ray absorption near edge structural (XANES) spectroscopy and extended X-ray absorption fine structural (EXAFS) spectroscopy. The extraction efficiency could be improved by increasing the temperature during extraction. The patterns of nanoparticles in IL-I during the extraction also changed. We identified the changes in nanoparticles which were suitable for reuse. The molecular-scale data were useful in revealing the process of copper and ITO separation from the scrap Cu/ITO thin film.Ionic liquids have unique properties, such as low vapor pressure, large liquid temperature ranges, and wide electrochemical windows. Ionic liquids are used in synthesis, electrochemistry, catalysis, carbon capture, and separation ,15,16,17and HCl) . The ionand HCl) . Anotherand HCl) . Moreoveand HCl) . Althougand HCl) . We usedhin film . In this2O3 was still maintained on the regenerated ITO thin film. The resistivity of the regenerated ITO thin film was 1.99 Ω.sq after extraction using a four-point probe. Note that the resistivity of unused ITO thin film was 1.98 Ω.sq. The Cu/ITO thin films can be regenerated by extracting copper species with the IL-I at 303–543 K.About 99% of the copper on the surface of the scrap Cu/ITO thin film was extracted into the IL-I at 303–543 K within 30 min see . At 473 xy electron transition, meaning the Cu(I) could be observed in the IL-I (see 2− (56%), CuI (39%), and Cu (5%) were the main species in the IL-I at 303 K . CuI was the main species on the Cu/ITO thin film at 303–543 K. Therefore, I3− interacted with Cu and then formed CuI on the surface of the scrap Cu/ITO thin film. Moreover, CuI stripped from the surface of scrap Cu/ITO thin film into the IL-I can interact with I− to form CuI2−. In XRD, CuI on the scrap Cu/ITO thin film was observed during extraction with IL-I at 303, 343, 413, 473, and 543 K, respectively  were less than 0.01 Å2   were added to 1 g of [C4mim][PF6] to form IL-I. The thickness of copper coated onto the surface of ITO thin films was approximately 380–400 nm. The Cu/ITO was collected from touch panels in Taiwan. It was estimated that 0.03 g of copper on the scrap Cu/ITO thin films was extracted into one g of IL-I from 303 K to 543 K within 30 min. The spectra of samples were collected at 303, 343, 413, 473, and 543 K during extraction. The samples after IL-I extraction were digested and the copper concentrations were then measured by AA . The extraction efficiencies of copper from the Cu/ITO to IL-I were estimated by the following equation:C1 (mg/g) is the initial concentration of copper in Cu/ITO and C2 (mg/g) is the concentration of extracted copper in IL-I.The synthesis process of − of IL-I could affect the growth of nanoparticles.Approximately 99% of copper on scrap Cu/ITO was extracted with the IL-I at 303–473 K. Using XRD, crystal In